pARA-R Recombinant
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pARA-R Recombinant RFP Expression Sequence biotechnology lab program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.1
description
biotechnology lab program. pARA-R Recombinant. RFP Expression Sequence. Laboratory Protocols by:. Marty Ikkanda. Powerpoint by:. Anthony Daulo. Pierce College, Woodland Hills, CA. V.1.2.1. restriction analysis of pKAN-R and pARA. pKAN-R. 5408 bp. Bam H I. rfp 702 bp. pARA. 4058 bp. - PowerPoint PPT Presentation
Transcript of pARA-R Recombinant
pARA-R RecombinantRFP Expression Sequence
biotechnology lab program
Laboratory Protocols by:Marty Ikkanda
Powerpoint by:Anthony Daulo
Pierce College, Woodland Hills, CA
V.1.2.1
restriction analysis of pKAN-R and pARA
pKAN-R
pARA
BamH I
Hind III
BamH IHind III
rfp702 bp
5408 bp
4058 bp
40 bp
restriction analysis of pKAN-R and pARA
BamH I Hind III
702bp
40 bp
BamH I Hind III
BamH IHind III
BamH IHind III
Restriction fragments after digest with Hind III and BamH I
4018 bp
4706 bp
restriction analysis of pKAN-R and pARA
Prediction for restriction gel
M K+ K- A+ A-M K+ K- A+ A-
500
1000
1500
20003000400050008000
10000
ligation of pKAN-R/pARA restriction fragments
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
ligation of pKAN-R/pARA restriction fragments
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
restriction analysis of pKAN-R and pARA
M K+ K- A+ A-
500
1000
1500
20003000400050008000
10000
Confirmation of restriction and ligation
L M K+ K- A+ A- L
preparing competent cells for transformation
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
Adhesion zone
Calcium ions
transforming Escherichia coli with pARA-R
Recombinant Plasmids
Competent Cells
pARA-R
Calcium ions
pARA-R
transforming Escherichia coli with pARA-R
Adhesion zone
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
growth of transformed bacteria on various plates
P+ plates
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
Colony isolation and culture
preparing an overnight culture of E. Coli for RFP expression
LB/amp/arabroth
RFP expression
araC gene rfp genePBAD
Transcription
mRNA
Translation
araC protein
RFP expression
rfp genePBAD
araC protein
araC gene
araC protein prevents RFP transcription by causinga loop to form in the region of the fp gener
RFP expression
araC protein
arabinose
araC gene rfp genePBAD
arabinose – araC proteincomplex
RNA polymerase
Arabinose – araC protein complex prevents DNA loopingand helps to align RNA polymerase
on the promoter site (PBAD).
mRNA
Transcription
TranslationRFP
(red fluorescentprotein)
RFP
purification of RFP from an overnight culture
Overnightculture
Cell pelletwith RFP
Lysedcells
Pelletcell debris
RFP withbinding buffer
